Method and system for inactivating virus infectivity for producing live-attenuated vaccines

ABSTRACT

Embodiments relate to methods comprising expressing or overexpressing P-selectin glycoprotein ligand-1 (PSGL-1) in human immunodeficiency virus (HIV) producing cells; isolating HIV particles from the HIV producing cells; and preparing the isolated HIV particles as a HIV vaccine. Embodiments relate to systems comprising a HIV vaccine comprising live attenuated, inactivated, or non-infectious HIV particles. Embodiments relate to systems capable of performing a method comprising administering a vaccine comprising live attenuated, inactivated, or non-infectious HIV particles to a subject in need of the vaccine; and treating or preventing one or more disease states in the subject resulting from HIV infection. Embodiments relate to methods comprising expressing or overexpressing PSGL-1 in virus producing cells; and inhibiting viral infection; or inhibiting viral spreading; or inactivating viruses and virus producing cells; or producing non-infectious virion particles; or allowing the virus producing cells to produce non-infectious virions, isolating the virions, and preparing non-infectious virions, the virions being HIV particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.16/271,100, filed on Feb. 8, 2019, which claims the benefit under 35U.S.C § 119 of U.S. Provisional Application No. 62/628,073, filed onFeb. 8, 2018, which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to the process of preparing effective vaccines,especially against viruses such as human immunodeficiency virus (HIV),comprising non-infectious, inactivated, and/or attenuated virionparticles. The invention is more particularly concerned with a processinvolving the expression or overexpression of a protein in a virusproducing host cell, such that the virion particles produced arenon-infectious, inactivated, and/or attenuated.

BACKGROUND OF INVENTION

An embodiment relates to preparing non-infectious virion particles byusing the expression or overexpression of a protein, such as P-selectinglycoprotein ligand-1 (PSGL-1), in virus producing cells to inactivateviral infectivity.

Currently, the preparation of live attenuated viral vaccines frequentlyrequires the inactivation of viral infectivity, resulting innon-infectious virion particles, using chemical and/or biologicalmethods, which have disadvantages. In the case of some viruses, such asHIV, there are no existing methods to prepare live, inactivated, and/orattenuated virus particles for use in vaccines.

Chemical methods for the preparation of inactivated viral vaccinesinclude treating isolated virus or virion particles with chemicalsubstances that tend to destroy the structure of the virus particles,resulting in altered immunogenicity of the virus. Thus, a personvaccinated with a vaccine based on chemically treated virus particlesmay not produce an immune response effective for the wild type virus. Inaddition, vaccines produced using chemical treatment of virus particlesmay be contaminated with chemical impurities that may be harmful topatients.

Biological methods of preparing live attenuated viral vaccines oftenrely on creating genetically modified or mutant virus particles thathave less infectivity than wild type viruses. However, in someinstances, such as smallpox, even such attenuated viruses may causesymptoms in subjects due to low-level viral replication, leading todisease states. Such biological methods require specific knowledge ofwhich gene mutations lead to a safe attenuated virus. For some viruses,such as HIV, such knowledge is not available. Thus, currently, there areno biological methods available to produce attenuated strains of HIV.

Until now, the expression or overexpression of proteins, such as PSGL-1,in virus producing cells to isolate virus particles from the virusproducing cells and prepare isolated virus particles, such as HIV virusparticles, as a virus vaccine has not been used in the preparation ofvaccines. Furthermore, such a method has not been used to produce virusparticles that are either non-infectious, attenuated, or inactivated.Expression or overexpression of proteins, such as PSGL-1, in virusproducing cells has not been used to inhibit viral infection, inhibitviral spreading, inactivate viruses and viral reservoirs, or producenon-infectious virion particles, such as HIV virion particles. Theexpression or overexpression of PSGL-1 in virus producing cells has notbeen performed by introducing a vector expressing PSGL-1 into the virusproducing cells. Also, a system comprising an HIV vaccine comprisinglive attenuated, inactivated, or non-infectious HIV particles has notbeen created. Nor has such a system been created, wherein the HIVparticles are produced in virus producing cells, in which PSGL-1 isexpressed or overexpressed. Furthermore, such systems have not been usedto administer a vaccine to a subject to treat or prevent one or moredisease states resulting from HIV infection.

SUMMARY OF INVENTION

An embodiment relates to a method, comprising: expressing P-selectinglycoprotein ligand-1 (PSGL-1) in human immunodeficiency virus (HIV)producing cells; isolating HIV particles from the HIV producing cells;and preparing the isolated HIV particles as a HIV vaccine.

In one embodiment, the HIV particles are non-infectious HIV particles.

In one embodiment, the HIV particles are attenuated HIV particles.

In one embodiment, the HIV particles are inactivated HIV particles.

In one embodiment, the method does not require a chemical that changes astructure of the HIV producing cells.

An embodiment relates to a method, comprising: expressing PSGL-1 invirus producing cells; and inhibiting virus producing cells fromproducing infectious viruses.

An embodiment relates a method, comprising: expressing PSGL-1 in virusproducing cells; and inhibiting viral spreading.

An embodiment relates to a method, comprising: expressing PSGL-1 invirus producing cells; and inactivating viruses.

An embodiment relates to a method, comprising: expressing PSGL-1 invirus producing cells; and producing non-infectious virion particles.

In one embodiment, PSGL-1 is overexpressed in the virus producing cells.

In one embodiment, PSGL-1 is expressed by introducing a vectorexpressing PSGL-1 into the virus producing cells.

In one embodiment, the virus producing cells produce HIV.

In one embodiment, the virus producing cells are host cells infectedwith the virus.

In one embodiment, the virus is HIV.

An embodiment relates to a method, comprising: expressing PSGL-1 invirus producing cells; allowing the virus producing cells to producevirions; isolating the virions; and preparing non-infectious virions.

In one embodiment, the virions are HIV particles.

An embodiment relates to a vaccine, comprising: a human immunodeficiencyvirus (HIV) vaccine comprising live attenuated, inactivated, ornon-infectious HIV particles.

An embodiment relates to a vaccine, comprising: a human immunodeficiencyvirus (HIV) vaccine comprising live attenuated, inactivated, ornon-infectious HIV particles, wherein the HIV particles are produced invirus producing cells, and wherein PSGL-1 is expressed in the virusproducing cells.

Additional embodiments relate to one or more methods comprising:administering the vaccines claimed herein to a subject in need thereof;and treating or preventing one or more disease states in the subjectresulting from HIV infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a -FIG. 1d show that PSGL-1 promotes HIV-1 virion release.

FIG. 2a -FIG. 2c show that PSGL-1 promotes the release of non-infectiousvirion particles.

FIG. 3a -FIG. 3c show that PSGL-1 is incorporated into virion particles.

FIG. 4a -FIG. 4b shows that virion incorporation of PSGL-1 preventsvirion attachment and entry.

FIG. 5 shows that PSGL-1 also inhibits viral entry through theendocytotic pathway.

FIG. 6a -FIG. 6b show that PSGL-1 inactivates HIV infectivity.

FIG. 7a -FIG. 7e show that HIV-1 infection down-regulates PSGL-1.

FIG. 8a -FIG. 8h show that PSGL-1 restricts HIV-1 infectivity whenexpressed in the virus-producer cell.

FIG. 9a -FIG. 9h show that Vpu and Nef antagonize PSGL-1

FIG. 10a -FIG. 10c show that PSGL-1 is incorporated into virions and itsexpression and incorporation alters viral protein composition in virion.

FIG. 11a -FIG. 11c show validation of PSGL-1 expression followingtransfection of HEK293T and HeLa JC.53 cells.

FIG. 12 shows HIV-1 dose-dependent downregulation of PSGL-1 in Jurkat Tcells.

FIG. 13 shows that PSGL-1 blocks the establishment of a spreading HIV-1infection.

FIG. 14 shows that PSGL-1 inactivates VSV-G pseudotyped HIV-1 virioninfectivity.

FIG. 15 shows that cell-free PSGL-1 does not inhibit HIV-1 infectivity.

FIG. 16 shows a comparison of PSGL-1 inhibition of HIV-1 (WT) and HIV-1(ΔNef) viral replication.

FIG. 17a -FIG. 17d show virion incorporation of PSGL-1.

DETAILED DESCRIPTION

Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, healthmonitoring described herein are those well-known and commonly used inthe art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. The nomenclatures used in connection with, and the proceduresand techniques of virology, immunology, vaccinology, and other relatedfields described herein are those well-known and commonly used in theart.

The following terms and phrases, unless otherwise indicated, shall beunderstood to have the following meanings.

The term PSGL-1 as used herein refers to P-selectin glycoproteinligand-1, which is a membrane protein that mediates the surfacetethering and rolling of Th1 T cells for tissue migration, and has beensuggested to be up-regulated by Th1 cytokines such as interferon gamma.PSGL-1, also known as SELPLG or CD162, is primarily expressed on thesurface of lymphoid and myeloid cells and binds to all three members ofthe selectin family of proteins, P-, E-, and L-selectin. PSGL-1 isup-regulated during inflammation to mediate leukocyte tethering androlling on the surface of the endothelium to promote leukocyte migrationinto inflamed tissues. In a mouse model of chronic viral infection,PSGL-1 has been reported to be an immune factor regulating T-cellcheckpoints. In addition, PSGL-1 serves as a receptor for enterovirus 71(EV71) infection of leukocytes. PSGL-1 has also been shown to be anINF-γ-regulated factor involved in Th1-mediated antiviral activity.During T-cell differentiation, culturing T cells in the Th1 cytokineINF-γ and IL-12 promoted PSGL-1 expression preferentially in theINF-γ-producing T-cell population, suggesting that PSGL-1 could be anINF-γ-regulated factor involved in Th1-mediated antiviral activity.PSGL-1 was reported to co-cluster with HIV-1 Gag at sites of assembly inthe T-cell uropod.

The term virus or virion as used herein refers to a submicroscopicinfectious agent that is unable to grow or reproduce outside a hostcell. It is non-cellular but consisting of a core of DNA or RNAsurrounded by a protein coat. A virus is a small parasite that cannotreproduce by itself. Once it infects a susceptible cell, however, avirus can direct the cell machinery to produce more viruses. Virus andvirion as used herein are synonymous.

The phrase virus particle or virion particle as used herein refers to acomplete infectious agent that consists of an RNA or DNA core with aprotein coat sometimes with external envelopes and that is theextracellular infective form of a virus.

The phrase native structure of an isolated HIV particle as used hereinrefers to the naturally occurring three-dimensional structure of an HIVparticle, as encoded by the genetic material of the virus.

The term HIV as used herein refers to human immunodeficiency virus.Infection of subjects with HIV can result in diseases, includingacquired immune deficiency syndrome (AIDS).

The term subject as used herein refers to a human individual. Thisindividual could be a patient requiring prophylaxis and/or medicaltreatment.

The phrase virus producing cell as used herein refers to cell that avirus has infected and whose cell machinery the virus can direct toproduce more viruses.

The phrase expressing or overexpressing PSGL-1 in virus producing cellsas used herein refers to causing the protein PSGL-1 to be synthesized bythe cell in larger quantities than would normally be expressed by thecell on its own.

The phrase isolating HIV particles as used herein refers to separatingHIV particles from other substances, such as the components of the virusproducing host cell, using methods such as chromatography.

The phrase non-infectious virus particle as used herein refers to avirus particle that has been rendered unable to infect a host cell andcause one or more effects, such as disease or death of the host cell.

The phrase attenuated virus particle as used herein refers to a virusparticle that has been weakened in its ability to infect a host cell andcause one or more effects, such as disease or death of the host cell.

The phrase inactivated virus particle as used herein refers to a virusparticle that has been rendered unable to infect a host cell and causeone or more effects, such as disease or death of the host cell.

The phrase inhibiting viral infection as used herein refers todecreasing somewhat or fully the ability of a virus to infect a hostcell and cause one or more effects, such as disease or death of the hostcell.

The phrase inhibiting viral spreading as used herein refers todecreasing somewhat or fully the production of more viruses eitherwithin our outside of a cell or organism.

The phrase inactivating viruses as used herein refers to renderingviruses unable to cause one or more effects in a host cell, such asdisease or death of the host cell.

The phrase producing non-infectious virion particles as used hereinrefers to creating a virus that is unable to infect a host cell andcause one or more effects in the cell, such as disease or death of thehost cell.

The phrase introducing a vector as used herein refers to the process ofcausing a cell to take up a piece of genetic material, often foreign tothe cell.

The phrase host cell as used herein refers to a cell which a virusinfects to reproduce itself, because a virus is a small parasite thatcannot reproduce by itself.

The phrase host cells infected with the virus as used herein refers to ahost cell that has been affected in its normal functioning by theattachment and/or internalization of a virus.

The phrase disease state as used herein refers to a disorder ofstructure or function of a human caused by a virus.

The phrase allowing a virus producing cell to produce virions as usedherein refers to enabling a cell to complete the process of a virusdirecting the cell's machinery to reproduce the virus fully.

An embodiment makes it possible to express or overexpress P-selectinglycoprotein ligand-1 (PSGL-1) in human immunodeficiency virus (HIV)producing cells; isolate HIV particles from the HIV producing cells; andprepare the isolated HIV particles as a HIV vaccine.

In another aspect, an embodiment features a system comprising a humanimmunodeficiency virus (HIV) vaccine comprising live attenuated,inactivated, or non-infectious HIV particles.

Yet another embodiment features a system comprising a humanimmunodeficiency virus (HIV) vaccine comprising live attenuated,inactivated, or non-infectious HIV particles, wherein the HIV particlesare produced in virus producing cells, and wherein PSGL-1 is expressedor overexpressed in the virus producing cells. Furthermore, the systemdoes not require a chemical that destroy a structure of the HIVproducing cells.

An embodiment relates to a system capable of performing a methodcomprising administering a vaccine comprising live attenuated,inactivated, or non-infectious HIV particles to a subject in need of thevaccine; and treating or preventing one or more disease states in thesubject resulting from HIV infection.

An embodiment relates to a system capable of performing a methodcomprising administering a vaccine comprising live attenuated,inactivated, or non-infectious HIV particles, wherein the HIV particlesare produced in virus producing cells, and wherein PSGL-1 is expressedor overexpressed in the virus producing cells, to a subject in need ofthe vaccine; and treating or preventing one or more disease states inthe subject resulting from HIV infection.

Yet another embodiment relates to a method, comprising expressing oroverexpressing PSGL-1 in HIV producing cells; isolating HIV particlesfrom the HIV producing cells; and preparing the isolated HIV particlesas a HIV vaccine. The HIV particles may be non-infectious, attenuated,or inactivated. Furthermore, the method does not require a chemical thatdestroy a structure of the HIV producing cells.

Another embodiment relates to a method, comprising expressing oroverexpressing PSGL-1 in virus producing cells; and inhibiting viralinfection, or inhibiting viral spreading, or inactivating viruses andvirus producing cells, or producing non-infectious virion particles.

A variation of the embodiment described immediately above is expressingor overexpressing PSGL-1 by introducing a vector expressing PSGL-1 intothe virus producing cells.

Another embodiment relates to a method, comprising expressing oroverexpressing PSGL-1 in virus producing cells; and inhibiting viralinfection, or inhibiting viral spreading, or inactivating viruses, orproducing non-infectious virion particles, wherein the virus producingcells produce HIV, or wherein the virus producing host cells are hostcells infected with the virus, or wherein the virus producing host cellsare host cells infected with the HIV virus.

Yet another embodiment relates to a method, comprising expressing oroverexpressing PSGL-1 in virus producing cells; allowing the virusproducing cells to produce non-infectious viral particles; isolating thevirions; and preparing non-infectious virions. These virions could beHIV particles.

In one embodiment PSGL-1 restricts HIV infection through a novel “killand release” mechanism, overexpression of PSGL-1 promotes virionrelease, and the released virions lose infectivity. PSGL-1 restricts HIVspreading through direct virion incorporation, which inactivates progenyvirion attachment and entry into CD4 T cells. PSGL-1 restricts HIVinfection through promoting the release of non-infectious virionparticles. Given that the released virion particles are not able toattach to target cells, these particles are likely endocytosed byantigen presenting cells, and processed as antigens for MHC class IIantigen presentation to stimulate anti-viral humoral immunity.

In another embodiment PSGL-1 inhibits the infectivity of VSV-Gpseudo-typed lentiviruses, demonstrating that PSLG-1 potentially hasbroad anti-viral activity against different viruses. PSGL-1 can be usedto inactivate viral infectivity for producing live-attenuated viralvaccines.

In another embodiment PSGL-1 restricts HIV infection through promotingthe release of non-infectious virion particles. Given that the releasednon-infectious virion particles are not able to attach to target CD4 Tcells, these particles are likely endocytosed by antigen presentingcells, and processed as antigens for MHC class II antigen presentationto stimulate anti-HIV humoral immunity.

In another embodiment PSGL-1 promotes the release of non-infectiousvirions (“kill and release”). The inactivation by PSGL-1 of virioninfectivity may occur through blocking the protease processing of virionproteins. Some of the most abundant cellular proteins in the virionparticles are actin and cofilin (10-15% and 2-10% of gag). HIV reversetranscriptase, nuclear capsid, and Nef have been shown to bind to actindirectly. It is possible that F-actin may serve as a scuffled protein toorganize the proper positioning of various virion proteins and theirprecursors. PSGL-1 may interfere with the organization of virionproteins and affect their proper processing by protease.

In yet another embodiment PSGL-1 (P-selectin glycoprotein ligand-1) is adimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, andL-selectins. PSGL-1 is primarily expressed on the surface of lymphoidand myeloid cells and is up-regulated during inflammation to mediateleukocyte tethering and rolling on the surface of the endothelium formigration into inflamed tissues. The presence of high levels of PSGL-1on T cells potently restricts HIV-1 infectivity; reducing the attachmentof virions to target cells and impairing infectivity in bothsingle-round and spreading infections. PSGL-1 is itself incorporatedinto HIV-1 particles. HIV-1 infection, and expression of Vpu and Nef,downregulate PSGL-1 from the cell surface, enabling the virus toantagonize PSGL-1-mediated restriction.

In another embodiment PSGL-1, also known as SELPLG or CD162, isprimarily expressed on the surface of lymphoid and myeloid cells andbinds to all three members of the selectin family of proteins, P-, E-,and L-selectin. PSGL-1 mediates leukocyte tethering and rolling on thesurface of endothelium to promote leukocyte migration into inflamedtissues. In a mouse model of chronic viral infection, PSGL-1 is animmune factor regulating T-cell checkpoints. In addition, PSGL-1 servesas a receptor for enterovirus 71 (EV71) infection of leukocytes.

In one embodiment PSGL-1 is an INF-γ-regulated factor involved inTh1-mediated antiviral activity. During T-cell differentiation,culturing T cells in the Th1 cytokine INF-γ and IL-12 promotes PSGL-1expression preferentially in the INF-γ-producing T-cell population,suggesting that PSGL-1 could be an INF-γ-regulated factor involved inTh1-mediated antiviral activity. PSGL-1 co-clusters with HIV-1 Gag atsites of assembly in the T-cell uropod.

In another embodiment PSGL-1, a mucin-like glycoprotein highly expressedon blood resting CD4 T cells, restricts HIV-1 virion infectivity. Inthis respect, PSGL-1-mediated restriction resembles that imposed by theApobec3G, SERINC5, MARCH, GBPS, and 90K proteins, which also targetvirion infectivity. In contrast to the Vpu-antagonized restrictionfactor BST2/tetherin that tethers virion particles to the cell surface,PSGL-1 does not inhibit virion release. However, PSGL-1-imprintedvirions lose the ability to attach to, and infect, target cells. PSGL-1is a remarkably potent inhibitor of HIV-1 infectivity; it almostcompletely inactivated WT HIV-1 particle infectivity at avector-to-proviral DNA ratio of 0.05:1 (FIG. 8e and FIG. 8f ). Giventhis high potency, it appears that HIV-1 uses two of its accessoryproteins, Vpu and Nef, to antagonize PSGL-1.

One of the embodiments provides novel insights into the ability of thehost cell to interfere with HIV-1 infection, and the biological functionof lentiviral accessory proteins. Further elucidation of the mechanismby which PSGL-1 restricts HIV-1 infection may offer new therapeuticstrategies for targeting HIV-1 replication.

The disclosed embodiments change the way in which vaccines comprisinglive attenuated, inactivated, or non-infectious virion particles, suchas HIV particles, are produced.

FIGS. 1a-1d illustrates that PSGL-1 promotes HIV-1 virion release. Todetermine possible effects of PSGL-1 expression on HIV infection,HEK293T cells were cotransfected with HIV (NL4-3) DNA (1000 ng) plus aPSGL-1 expressing vector, using a range of vector dosages from 0.5 to800 ng. At low dosages (0.5 to 50 ng), PSGL-1 greatly promoted virionrelease (200 to 400% based on p24). At higher dosages (50 ng and above),the enhancement of virion release was decreased. The empty vector DNAwas added during transfection to maintain the same amount of DNA usedfor cotransfection. (FIGS. 1a and 1b ). However, when intracellularviral proteins were examined, PSGL-1 was found to inhibit intracellularp24 accumulation at dosages higher than 50 ng, and 800 ng PSGL-1 wastoxic to cells. Cotransfected cells and extracellular virion particleswere lyszed and analyzed by SDS-PAGE and western blot using ananti-PSGL-1 antibody and an anti-HIV antibody. (FIG. 1c ). Whennormalized to intracellular p24, PSGL-1 enhanced virion release at allnon-toxic dosages tested. The relative ratio of p24 on the western blotwas quantified (extracellular p24/intracellular p24). (FIG. 1d ). Theseresults appear to be consistent with an earlier report that PSGL-1 isinvolved in HIV assembly and budding. It is possible that increasing theamount of PSGL-1 to the sites of HIV assembly promotes virion release.

FIGS. 2a-2c illustrate that PSGL-1 promotes the release ofnon-infectious virion particles. The infectivity of the released virionto infect target CD4 T cells was examined using an HIV Rev-dependent GFPreporter cell line, A3R5-GFP-RRE. Unlike LTR-driving reporter cells, theRev-dependent report cell strictly requires HIV Rev to turn on GFPexpression, which is not affected by cellular factors present in thesupernatant of transfected HEK293T cells. Surprisingly, while

PSGL-1 promotes virion release, the particles released completely lostinfectivity at PSGL-1 vector dosages of 50 ng and higher (HIV: PSGL-1vector ratio, 1:0.05). HIV particles are also partially inactivated byPSGL-1 at dosages as low as 1-5 ng, and there is a dosage-dependentinactivation of HIV by PSGL-1 at dosages between 1 to 50 ng. HEK293Tcells were cotransfected with HIV (NL4-3) DNA (1 ug) plus 400 ng ofPSGL-1 expressing vector (PSGL-1). The empty vector DNA (vector) wasadded during transfection to maintain the same amount of DNA used forcotransfection. At 48 hours post co-transfection, virion particles wereharvested and used to infect an HIV Revdependent reporter cellsA3R5-GFP-RRE. GFP expression was quantified at 48 hours post infection.An equal p24 level of p24 was used for infection (FIGS. 2a and 2c ).Cotransfection and infection were done as in FIG. 2a ) and FIG. 2c ). Anequal volume of viruses were used the infection of A3R5-GFP-RRE (FIG. 2b).

FIG. 3 illustrates that PSGL-1 is incorporated into virion particles. Todetermine whether PSGL-1 is incorporated into HIV virion particles, wecotransfected HIV (NL4-3) DNA with the PSGL-1 vector, and purified thevirion particles by 2 rounds of high-speed centrifugation, through agradient of 6-18% OpiPrep solution. HEK293T cells were cotransfectedwith HIV (NL4-3) DNA (1 ug) plus 200 ng of a PSGL-1 expressing vector(PSGL-1). The empty vector DNA (vector) was added during transfection tomaintain the same amount of DNA used for cotransfection. Virions wereharvested at 48 hours post cotranfection and purified by ultra-speedcentrifugation in OptiPrep gradient solution (1.2-18%). (FIG. 3a ).Highly purified virion pellets were analyzed by SDS-PAGE Western blotusing an anti-PSGL-1 antibody. As shown in FIG. 3b , we detected thepresence of PSGL-1 in virion particles when the blot was probed with ananti-PSGL-1 antibody. In addition, we also probed the blot with anti-HIVantibodies, and detected the presence of various virion proteins.However, the relative ratios of virion proteins were altered. The polp66/p51/p31, and the p6/p7 proteins were diminished. Virion pellets wereanalyzed by SDS-PAGE and anti-HIV anti-serum (FIG. 3c ). These resultssuggest that the incorporation of PSGL-1 may affect the intravirionprotease cleavage of virion polyproteins, particularly the gag-polprecursors, which may block the maturation of released virion particles.

FIGS. 4a-4b illustrate that virion incorporation of PSGL-1 preventsvirion attachment and entry. The mechanism of PSGL-1-mediatedinactivation of virion infectivity was further studied by following theearly steps of HIV infection. Using an HIV entry assay, the BlaM-Vprassay, it was observed that the virions produced from PSGL-1vectorcotransfected cells were not able to enter CD4 T cells, suggestingthat virion incorporation of PSGL-1 may affect virion attachment orvirus-cell fusion. HEK293T cells were cotransfected with HIV (NL4-3) DNA(1 ug) plus 200 ng of a PSGL-1 expressing vector (PSGL-1). The emptyvector DNA (vector) was added during transfection to maintain the sameamount of DNA used for cotransfection. Virions were harvested at 48hours post cotranfection and used to perform a BlaM-Vprbased HIV entryassay. AMD3100 was used as a control (FIG. 4a ). A virion attachmentassay was also performed, and it was observed that the virions producedfrom PSGL-1 vector-cotransfected cells were not able to attach to HIVtarget cells. Virions were also used to perform a virus attachmentassay. Viruses were incubated with target CD4+CXCR4+ JC.53 cells at 4degree for 2 hours. Cells were extensively washed, lyzed, and analyzedby SDS-PAGE and western blot using an anti-HIV p24 antibody. The inputviruses used for the attachment assay were also analyzed. (FIG. 4b ).

FIG. 5 illustrates that PSGL-1 also blocks the infectivity of VSV-Gpseudo-typed HIV virion particles and inhibits viral entry through theendocytotic pathway. It was investigated whether PSGL-1-mediatedinhibition of viral entry is specific to the HIV envelope-mediatedplasma membrane fusion. HIV was pseudo-typed with the VSV-G envelope(vesicular stomatitis virus glycoprotein), which mediates viral entrythrough endocytosis rather than member fusion. As shown in FIG. 5, theinhibition of VSV-G pseudo-typed HIV infection was observed,demonstrating that PSGL-1 can block viral entry through both plasmamember fusion and endocytosis. PSGL-1 is expected to block theinfectivity of multiple viruses. HEK293T cells were cotransfected withHIV (KFS)+pCMV-VSV-G DNA plus a PSGL-1 expressing vector (PSGL-1) or theempty vector DNA (Empty). At 48 hours post co-transfection, virionparticles were harvested and used to infect an HIV Rev-dependentreporter cells A3R5-GFP-RRE. GFP expression was quantified at 48 hourspost infection.

FIGS. 6a-6b illustrate that PSGL-1 inactivates HIV infectivity. HEK293Tcells were cotransfected with HIV (NL4-3) DNA (1 ug) plus 200 ng of aPSGL-1 expressing vector (PSGL-1) or the empty vector (Vector). At 48hours post cotransfection, virion particles were harvested and used toinfect A3R5.7 CD4 T cells or A3R5-GFP-RRE. An equal p24 level of p24 wasused for infection. Viral replication was monitored by p24 release (FIG.6a ). PSGL-1 tranfection supernatant did not inhibit HIV infection.HEK293T cells were transfected with 200 ng of a PSGL-1 expressing vector(PSGL-1) or the empty vector (Vector). Supernatants were harvested at 48hours, and 400 ul of the supernatants were mixed with 200 ul of HIV-1(NL4-3) virion particles, and then used to infect A3R5-GFP-RRE. Forcontrol, 400 ul fresh medium was mixed with HIV (FIG. 6b ). The completeinactivation of HIV infectivity was confirmed by quantifying HIVspreading infection in A3R5 CD4 T cells by p24 release This inactivationof HIV infectivity was not caused by possible cellular factors presentin the cotransfection supernatant; the supernatant from PSGL-1vector-only transfected cells did not inhibit HIV infectivity when mixedwith HIV virion particles. Given that PSGL-1 is a membrane protein andis recruited to the sites of virion assembly, it is possible that PSGL-1may inactivate virion infectivity through direct incorporation intovirion particles.

FIGS. 7a-7e illustrates that HIV-1 infection down-regulates PSGL-1.(FIG. 7a ) Blood resting CD4 T cells were purified by negativeselection, activated with PHA+IL-2, or left unstimulated. Cell surfacePSGL-1 expression was analyzed by flow cytometry. (FIG. 7b ) Jurkat andCEM-SS cells were similarly stained for surface PSGL-1. (FIG. 7c ) Bloodresting CD4 T cells were infected with NLHG1-ES-IRES-GFP reporter virus(125 to 320 ng p24 per million cells). Following infection, cells werewashed and cultured in complete medium plus IL-7 (2 ng/ml) to permitlow-level viral replication. Surface PSGL-1 expression was analyzed atthe indicated days. Shown are the percentages of the GFP+ or GFP− cellswith low or high PSGL-1 staining in each panel. PSGL-1 down-regulationwas observed only in the HIV+/GFP+ cell population. (FIG. 7d ) Forcontrols, uninfected cells were similarly cultured in IL-7 and surfacePSGL-1 expression was analyzed at the indicated days. Culturing restingCD4 T cells in IL-7 did not lead to PSGL-1 downregulation. (FIG. 7e ),Cells were also stained for surface PSGL-1 expression on the CD45RA+(naïve) and CD45RA− (memory) CD4 T cells at day 7, and analyzed by flowcytometry.

FIGS. 8a-8h illustrates that PSGL-1 restricts HIV-1 infectivity whenexpressed in the virus-producer cell. (FIG. 8a and FIG. 8b ) HeLa JC.53cells were transfected with a PSGL-1 vector or a control empty vectorfor 48 hours, and then infected with HIV-1 (NL4-3) for spreadinginfection (FIG. 8a ) or HIV-1 (gp160) for single-round infection (FIG.8b ). Viral replication was quantified by p24 release at 72 hours (FIG.8a ) or 48 hours for (FIG. 8b ). (FIGS. 8c to 8f ) HEK293T cells werecotransfected with HIV (NL4-3) DNA (1 μg) plus different amounts ofPSGL-1 expression vector. Viral p24 release was quantified at 48 hours(FIG. 8c ). Cells were also lysed and analyzed by western blot forintracellular PSGL-1 and HIV-1 proteins (FIG. 8d ). Extracellular virionp24 was also analyzed by western blot (FIG. 8e ), and the relative ratioof extracellular and intracellular p24 was plotted (FIG. 8f ). (FIG. 8gand FIG. 8h ) Virions released from HEK293T cells cotransfected with HIV(NL4-3) DNA (1 μg) plus PSGL-1 DNA (0.5 to 400 ng) were harvested andnormalized for p24, and viral infectivity was quantified by infectingthe T-cell line-derived Rev-A3R5-GFP indicator cells. HIV-1 replicationwas quantified by GFP expression. Shown are the percentages of GFP+cells at 48-72 hours postinfection. The PSGL-1 dose-dependent inhibitioncurve in (FIG. 8h ) was plotted using results from 3 independentexperiments.

FIGS. 9a-9h illustrates that Vpu and Nef antagonize PSGL-1. (FIG. 9a andFIG. 9b ) Downregulation of PSGL-1 from the cell surface by Vpu and Nef.HEK293T cells were cotransfected with PSGL-1 (100 ng) and a Vpu (FIG. 9a), or Nef (FIG. 9b ) expression vector at various DNA inputs. SurfacePSGL-1 expression was quantified and shown as the percentages of cellsexpressing PSGL-1. For controls, an empty vector was used (+Vector). Thesame amount of DNA was used in all transfections. (FIG. 9c and FIG. 9d )Levels of intracellular PSGL-1 in Vpu- or Nef-cotransfected cells werequantified by western blot at 48 hours post-cotransfection. (FIG. 9e andFIG. 9f ) Vpu and Nef antagonize PSG1-1. HEK293T cells werecotransfected with various amounts of PSGL-1 DNA (0.5-50 ng) plus 1 μgHIV (NL4-3) WT, HIV (ΔVpu), or HIVΔNef DNA. Virions were harvested andused to infect Rev-A3R5-GFP indicator cells. GFP expression wasquantified and shown as the percentage of GFP+ cells at 72 hourspostinfection. (FIG. 9g and FIG. 9h ) HeLa JC.53 cells were stablytransfected with PSGL-1 or empty vector DNA and drug-selected to obtainstably transfected cells. Cells were then infected with 3 differentinputs of HIV-1 (NL4-3) WT, HIV-1 (ΔVpu) (FIG. 9g ), or HIV-1 (ΔNef)(FIG. 9h ). Viral replication was quantified by p24 release. For (FIG.9h ), only the data for 32 ng p24 input at day 10 are shown.

FIGS. 10a-10c illustrate that PSGL-1 is incorporated into virions andits expression alters viral protein composition in virion particles.(FIG. 10a ) HEK293T cells were cotransfected with PSGL-1 DNA plus HIV-1(NL4-3). Virion particles were harvested at 48 hourspost-cotransfection, and intracellular and virion proteins were analyzedby western blot using antibodies against PSGL-1 (polyclonal) or HIVproteins (anti-HIV serum). The red stars highlight the severe reductionsin protein levels in in virions produced from PSGL-1-expressing cells.Positions of the Gag precursor protein (pr55) and p24 (CA) areindicated. (FIG. 10b ) Virions produced from HEK293T cells cotransfectedwith PSGL-1 expression vector plus HIV-1 (NL4-3) were used for an entryassay. As controls, HIV-1 virions similarly produced in the presence ofan empty vector were used. Equal p24 was used for the assay. The entryinhibitor AMD3100 was also used as a control to block virus entry. Thepercentages of cells with cleaved CCF2 are shown. FIG. 10c , Virionparticles produced in the presence of PSGL-1 or the empty vector wereassayed for attachment to target HeLa JC.53 cells at 4° C. for 2 hours.Cells were washed and then analyzed by western blot for cell-associatedp24.

FIGS. 11a-11c illustrate the Validation of PSGL-1 expression followingtransfection of HEK293T and HeLa JC.53 cells. (FIG. 11a and FIG. 11b ),HEK293T cells were transfected with a PSGL-1 expression vector(pCMV-PSGL-1), and then analyzed by western blot using 3 differentcommercial antibodies (FIG. 11a ). Expression of PSGL-1 on the surfacewas analyzed by surface staining and flow cytometry. Shown are thepercentages of cells with high or low PSGL-1 staining in each panel.(FIG. 11c ) HeLa JC.53 cells were transfected with pCMV-PSGL-1 at theindicated inputs (ng). PSGL1 expression was analyzed by western blot at48 hours posttransfection using anti-PSGL-1 polyclonal antibodies. GAPDHwas similarly analyzed as a loading control.

FIG. 12 illustrates the HIV-1 dose-dependent downregulation of PSGL-1 inJurkat T cells. Jurkat T cells were infected with different inputs ofHIV-1, washed and cultured for 3 days, and then stained for surfacePSGL-1 expression, and analyzed by flow cytometry. Shown are thepercentages of cells with high or low PSGL-1 staining in each panel.

FIG. 13 illustrates that PSGL-1 blocks the establishment of a spreadingHIV-1 infection. HEK293T cells (3×106) were cotransfected with 12 μg ofHIV (NL4-3) plus 2.4 μg pCMV-PSGL-1 or an empty vector. Viruses wereharvested at 48 hours post-transfection and used to infect A3R5.7 CD4 Tcells. After infection for 4 hours, cells were washed and cultured for 5days. HIV replication was analyzed by p24 release.

FIG. 14 illustrates that PSGL-1 inactivates VSV-G pseudotyped HIV-1virion infectivity. HEK293T cells (2×105) were cotransfected with 1 μgNL4-3 (KFS), 1 μg pHCMV-G, and 200 ng pCMV-PSGL-1 or an empty vector.Virus supernatants were harvested at 48 hours posttransfection, and usedto infect Rev-A3R5-GFP cells. After infection for 4 hours, cells werewashed and cultured in medium for 72 hours. The percentages of infected(GFP+) cells were quantified by flow cytometry.

FIG. 15 illustrates that Cell-free PSGL-1 does not inhibit HIV-1infectivity. HEK293T cells (2×105) were cotransfected with 200 ng ofpCMV-PSGL-1 or empty vector. Supernatants were collected at 48 hoursposttransfection and mixed with HIV (NL4-3) virus. The mixture was usedto infect Rev-A3R5-GFP for 4 hours. Following infection, cells werewashed and cultured for 72 hours. The percentages of infected (GFP+)cells were quantified by flow cytometry.

FIG. 16 illustrates the Comparison of PSGL-1 inhibition of HIV-1 (WT)and HIV-1 (ΔNef) viral replication. HeLa JC.53 cells were stablytransfected with PSGL-1 or empty vector DNA, and drug-selected to obtainstably transfected cells. Cells were then infected with 3 differentinputs of HIV-1 (NL4-3) WT or HIV-1 (ΔNef). Viral replication wasquantified by p24 release.

FIGS. 17a-17d illustrate the Virion incorporation of PSGL-1. (FIG. 17ato FIG. 17c ) HEK293T cells were cotransfected with varying amounts ofPSGL-1 DNA plus 1 μg HIV-1 (NL4-3) or HIV (ΔVpu) DNA at the indicatedratios. Virion particles were harvested at 48 hours and purified by tworounds of ultra-speed centrifugation through an OptiPrep gradient.Virion proteins were analyzed by western blot using antibodies againstPSGL-1 (polyclonal) or HIV-1 proteins (anti-HIV serum). Positions of Gagprecursor protein (pr55) and p24 (CA) are indicated. (FIG. 17d ) Forcontrols, HEK293T cells were transfected with PSGL-1 DNA only (200 ng)or co-transfected with HIV-1 DNA (1 μg) plus PSGL-1 DNA (200 ng).Extracellular vesicles or virion particles were similarly pelleted byultracentrifugation, and then analyzed by western blot for the presenceof PSGL-1 using the polyclonal antibody.

EXAMPLE 1

To determine possible effects of PSGL-1 expression on HIV infection,HEK293T cells were cotransfected with HIV (NL4-3) DNA (1000 ng) plus aPSGL-1 expressing vector, using a range of vector dosages from 0.5 to800 ng. At low dosages (0.5 to 50 ng), PSGL-1 greatly promoted virionrelease (200 to 400% based on p24). At higher dosages (50 ng and above),the enhancement of virion release was decreased. The empty vector DNAwas added during transfection to maintain the same amount of DNA usedfor cotransfection. However, when intracellular viral proteins wereexamined, PSGL-1 was found to inhibit intracellular p24 accumulation atdosages higher than 50 ng, and 800 ng PSGL-1 was toxic to cells.Cotransfected cells and extracellular virion particles were lyszed andanalyzed by SDS-PAGE and western blot using an anti-PSGL-1 antibody andan anti-HIV antibody. When normalized to intracellular p24, PSGL-1enhanced virion release at all non-toxic dosages tested. The relativeratio of p24 on the western blot was quantified (extracellularp24/intracellular p24). These results appear to be consistent with anearlier report that PSGL-1 is involved in HIV assembly and budding. Itis possible that increasing the amount of PSGL-1 to the sites of HIVassembly promotes virion release.

EXAMPLE 2

The infectivity of the released virion to infect target CD4 T cells wasexamined using an HIV Rev-dependent GFP reporter cell line,A3R5-GFP-RRE. Unlike LTR-driving reporter cells, the Rev-dependentreport cell strictly requires HIV Rev to turn on GFP expression, whichis not affected by cellular factors present in the supernatant oftransfected HEK293T cells. Surprisingly, while PSGL-1 promotes virionrelease, the particles released completely lost infectivity at PSGL-1vector dosages of 50 ng and higher (HIV: PSGL-1 vector ratio, 1:0.05).HIV particles are also partially inactivated by PSGL-1 at dosages as lowas 1-5 ng, and there is a dosage-dependent inactivation of HIV by PSGL-1at dosages between 1 to 50 ng. HEK293T cells were cotransfected with HIV(NL4-3) DNA (1 ug) plus 400 ng of PSGL-1 expressing vector (PSGL-1). Theempty vector DNA (vector) was added during transfection to maintain thesame amount of DNA used for cotransfection. At 48 hours postco-transfection, virion particles were harvested and used to infect anHIV Revdependent reporter cells A3R5-GFP-RRE. GFP expression wasquantified at 48 hours post infection. An equal p24 level of p24 wasused for infection. Cotransfection and infection were done as before. Anequal volume of viruses were used the infection of A3R5-GFP-RRE.

EXAMPLE 3

To determine whether PSGL-1 is incorporated into HIV virion particles,we cotransfected HIV (NL4-3) DNA with the PSGL-1 vector, and purifiedthe virion particles by 2 rounds of high-speed centrifugation, through agradient of 6-18% OpiPrep solution. HEK293T cells were cotransfectedwith HIV (NL4-3) DNA (1 ug) plus 200 ng of a PSGL-1 expressing vector(PSGL-1). The empty vector DNA (vector) was added during transfection tomaintain the same amount of DNA used for cotransfection. Virions wereharvested at 48 hours post cotranfection and purified by ultra-speedcentrifugation in OptiPrep gradient solution (1.2-18%). Highly purifiedvirion pellets were analyzed by SDS-PAGE Western blot using ananti-PSGL-1 antibody. We detected the presence of PSGL-1 in virionparticles when the blot was probed with an anti-PSGL-1 antibody. Inaddition, we also probed the blot with anti-HIV antibodies, and detectedthe presence of various virion proteins. However, the relative ratios ofvirion proteins were altered. The pol p66/p51/p31, and the p6/p7proteins were diminished. Virion pellets were analyzed by SDS-PAGE andanti-HIV anti-serum. These results suggest that the incorporation ofPSGL-1 may affect the intravirion protease cleavage of virionpolyproteins, particularly the gag-pol precursors, which may block thematuration of released virion particles.

EXAMPLE 4

The mechanism of PSGL-1-mediated inactivation of virion infectivity wasfurther studied by following the early steps of HIV infection. Using anHIV entry assay, the BlaM-Vpr assay, it was observed that the virionsproduced from PSGL-1 vectorcotransfected cells were not able to enterCD4 T cells, suggesting that virion incorporation of PSGL-1 may affectvirion attachment or virus-cell fusion. HEK293T cells were cotransfectedwith HIV (NL4-3) DNA (1 ug) plus 200 ng of a PSGL-1 expressing vector(PSGL-1). The empty vector DNA (vector) was added during transfection tomaintain the same amount of DNA used for cotransfection. Virions wereharvested at 48 hours post cotranfection and used to perform aBlaM-Vprbased HIV entry assay. AMD3100 was used as a control. A virionattachment assay was also performed, and it was observed that thevirions produced from PSGL-1 vector-cotransfected cells were not able toattach to HIV target cells. Virions were also used to perform a virusattachment assay. Viruses were incubated with target CD4+CXCR4+ JC.53cells at 4 degree for 2 hours. Cells were extensively washed, lyzed, andanalyzed by SDS-PAGE and western blot using an anti-HIV p24 antibody.The input viruses used for the attachment assay were also analyzed.

EXAMPLE 5

We investigated whether PSGL-1-mediated inhibition of viral entry isspecific to the HIV envelope-mediated plasma membrane fusion. HIV waspseudo-typed with the VSV-G envelope (vesicular stomatitis virusglycoprotein), which mediates viral entry through endocytosis ratherthan member fusion. The inhibition of VSV-G pseudo-typed HIV infectionwas observed, demonstrating that PSGL-1 can block viral entry throughboth plasma member fusion and endocytosis. PSGL-1 is expected to blockthe infectivity of multiple viruses. HEK293T cells were cotransfectedwith HIV (KFS)+pCMV-VSV-G DNA plus a PSGL-1 expressing vector (PSGL-1)or the empty vector DNA (Empty). At 48 hours post co-transfection,virion particles were harvested and used to infect an HIV Rev-dependentreporter cells A3R5-GFP-RRE. GFP expression was quantified at 48 hourspost infection.

EXAMPLE 6

HEK293T cells were cotransfected with HIV (NL4-3) DNA (1 ug) plus 200 ngof a PSGL-1 expressing vector (PSGL-1) or the empty vector (Vector). At48 hours post cotransfection, virion particles were harvested and usedto infect A3R5.7 CD4 T cells or A3R5-GFP-RRE. An equal p24 level of p24was used for infection. Viral replication was monitored by p24 release.PSGL-1 tranfection supernatant did not inhibit HIV infection. HEK293Tcells were transfected with 200 ng of a PSGL-1 expressing vector(PSGL-1) or the empty vector (Vector). Supernatants were harvested at 48hours, and 400 ul of the supernatants were mixed with 200 ul of HIV-1(NL4-3) virion particles, and then used to infect A3R5-GFP-RRE. Forcontrol, 400 ul fresh medium was mixed with HIV. The completeinactivation of HIV infectivity was confirmed by quantifying HIVspreading infection in A3R5 CD4 T cells by p24 release This inactivationof HIV infectivity was not caused by possible cellular factors presentin the cotransfection supernatant; the supernatant from PSGL-1vector-only transfected cells did not inhibit HIV infectivity when mixedwith HIV virion particles. Given that PSGL-1 is a membrane protein andis recruited to the sites of virion assembly, it is possible that PSGL-1may inactivate virion infectivity through direct incorporation intovirion particles.

EXAMPLE 7

To study the potential effects of PSGL-1 expression on HIV-1replication, we quantified PSGL-1 expression following HIV-1 infectionof primary resting CD4 T cells and the transformed Jurkat T-cell line.We observed that human blood resting CD4 T cells express high levels ofPSGL-1, and T-cell activation (IL-2 plus PHA) down-regulates PSGL-1,while transformed CD4 T-cell lines express low (e.g. Jurkat) toundetectable (e.g. CEM-SS) levels of PSGL-1. We found that HIV-1infection of IL-7-treated blood resting CD4 T cells down-regulatesPSGL-1 exclusively in the HIV-1+ cell population; culturing resting CD4T cells in IL-7 permits low-level HIV replication. HIV-1-mediateddownregulation of PSGL-1 occurred both in the memory and naïve T-cellpopulations. We also observed an HIV-1 dose-dependent downregulation ofPSGL-1 in infected Jurkat T cells. Thus, HIV-1 actively down-regulatessurface PSGL-1 on both primary resting memory and naïve CD4 T cells, aswell as on transformed T-cell lines.

EXAMPLE 8

Given the observed PSGL-1 down-regulation by HIV-1, we investigated therole of PSGL-1 in HIV-1 infection and spread. We transiently transfectedHeLa JC.53 cells with a PSGL-1 vector (5-50 ng), and then infected thecells with HIV (NL4-3) or an HIV-1 envelope (Env)-pseudotyped,single-cycle virus, HIV (gp160). We observed a dose-dependent inhibitionof HIV (NL4-3) in a multi-round, spreading infection, but not in asingle-cycle infection. Thus, at these low doses (5-50 ng), PSGL-1 didnot block any steps in the viral replication cycle up to the release ofvirion particles but rather blocked the establishment of a spreadinginfection.

EXAMPLE 9

To investigate the effects of PSGL-1 on late steps of the virusreplication cycle, we co-transfected HEK293T cells with HIV (NL4-3) DNA(1 μg) plus PSGL-1 expression vector at varying inputs from 0.5 to 800ng. We observed small effects of PSGL-1 expression on HIV-1 virionrelease, from slight enhancement at low doses (below 100 ng) to slightinhibition at high doses (800 ng). However, when normalized to thelevels of intracellular Gag, PSGL-1 did not inhibit virion release atany dose tested. Next, we quantified the infectivity of the releasedvirions on target CD4 T cells, using a highly stringent Rev-dependentindicator cell line, Rev-A3R5-GFP, that does not respond tonon-infectious HIV stimuli as do LTR-based reporter cell lines. We foundthat while PSGL-1 did not inhibit virion release, it abolished theinfectivity of released virions at PSGL-1 vector doses higher than 50ng. PSGL-1 partially restricted HIV-1 infectivity at inputs as low as0.5 ng (PSGL-1 DNA to HIV DNA ratio, 1:2000), and there was adose-dependent inactivation of HIV-1 at PSGL-1 vector doses from 0.5 to50 ng. The complete inactivation of HIV-1 infectivity was confirmed byquantifying HIV-1 spreading infection in A3R5.7 CD4 T cells by p24release. In addition, PSGL-1 expression in the virus-producer cell alsoblocked the infectivity of VSV-G pseudotyped HIV-1. This inactivation ofvirus infectivity was not caused by soluble PSGL-1 or PSGL-1-containingvesicles present in the cotransfection supernatant, as the supernatantfrom cells transfected with only PSGL-1 did not inhibit HIV-1infectivity when mixed with virion particles produced fromPSGL-1-negative HEK293T cells. Together, these results demonstrate thatthe presence of PSGL-1 in producer cells inactivates the infectivity ofreleased virions.

EXAMPLE 10

We demonstrated that HIV-1 antagonizes PSGL-1 by activelydown-regulating it from the T-cell surface. To identify the viralfactors responsible for HIV-1-mediated PSGL-1 downregulation, wecotransfected PSGL-1 DNA with Vpu or Nef expression vectors. Both Vpuand Nef are known broad-spectrum modulators of cell-surface receptors.We observed a dose-dependent downmodulation of PSGL-1 levels at the cellsurface by both Vpu and Nef, consistent with previous reports. However,when the levels of intracellular PSGL-1 were examined, Vpu but not Nefwas found to cause a decrease in total intracellular PSGL-1.Interestingly, Nef induced an intracellular accumulation of the 70-80kDa species of PSGL-1, suggesting that this low-mobility PSGL-1 may bethe form redirected by Nef to intracellular vesicles without beingdegraded. To confirm the biological role of Vpu and Nef in antagonizingPSGL-1, we cotransfected varying amounts of PSGL-1 DNA with wild-typeHIV-1 (NL4-3) DNA (WT) or mutants lacking Vpu (HIV-1ΔVpu) or Nef(HIVΔNef) expression. Virions were harvested and their infectivity wasquantified on T-cell line-derived Rev-A3R5-GFP indicator cells. For WTHIV-1, we observed a PSGL-1 dose-dependent inhibition of HIV-1infectivity, with 50% inhibitory dose (IC50) of around 2.71 ng ofPSGL-1. For HIV-1ΔVpu, we observed a similar dose-dependent inhibition,but there was a 10-fold difference in the sensitivity to PSGL-1restriction. The IC50 of PSGL-1 for HIVΔVpu is 0.29 ng. Thus, deletionof Vpu led to a heightened sensitivity to PSGL-1 restriction. There wasonly a slight difference in IC50s between HIVΔNef and HIV WT. Thus,although both Vpu and Nef can down-regulate PSGL-1 expression from thecell surface, Vpu appears to play the major role in antagonizing PSGL-1in HEK293T cells. These results were corroborated in spreading infectionof PSGL-1-stably transfected HeLa JC.53 cells (HeLaJC53-PSGL-1). WTHIV-1 and HIVΔVpu or HIVΔNef derivatives were produced in HEK293T cellsin the absence of PSGL-1, and then used to infect HeLaJC53-PSGL-1 orcontrol HeLaJC53-Empty vector-transfected cells, using p24-normalizedinocula. At all 3 HIV-1 inputs, PSGL-1 displayed a stronger inhibitionof HIVΔVpu than of WT. At the highest HIV-1 input used (32 ng p24), nospreading viral replication was detected from HIVΔVpu in HeLaJC53-PSGL-1cells, whereas WT viral replication was only modestly inhibited. Incomparison with HIVΔVpu, HIVΔNef was inhibited to a similar degree asthe HIV (WT) by PSGL-1. Based on these results, we conclude thatalthough both Vpu and Nef can downregulate PSGL-1 from the surface, Vpuplays the major role in antagonizing PSGL-1. Nevertheless, it is likelythat both viral factors work together in a cooperative manner toantagonize PSGL-1.

EXAMPLE 11

PSGL-1 has been found to co-localize with Gag at sites of HIV-1 Gagassembly in uropod microdomains following Gag multimerization. However,the biological role of PSGL-1/Gag co-localization during HIV virionassembly remains unclear. Given the reported colocalization of Gag andPSGL-1 in virus-producing cells, we asked whether PSGL-1 is incorporatedinto HIV-1 particles. We co-transfected HIV (NL4-3) DNA with the PSGL-1expression vector, purified the virion particles by two rounds ofultracentrifugation through an OptiPrep gradient, and analyzed thevirion content by western blot. We detected the presence of PSGL-1 invirions. Strikingly, levels of the viral Env glycoprotein subunits gp120and gp41 were significantly diminished when PSGL-1 was expressed in theproducer cell. We also observed a defect in Env precursor gp160processing to gp120 and gp41 in PSGL-1-expressing cells. Consistent withthe reduction in virion-associated Env proteins, we observed diminishedvirus entry when the PSGL-1-imprinted HIV-1 particles were used toperform a virus entry assay. We further performed a virion attachmentassay, and observed that virions from PSGL-1-expressing cells wereimpaired in their ability to attach to susceptible target cells.

Methods

Examples 7 through 11 relied on the use of the processes and materialsdescribed below.

Cells and Viruses

Peripheral blood buffy coats from HIV-1-negative adults were purchasedfrom the New York Blood Center or received from the NIH Blood Bank. CD4+T cells were isolated by negative selection using the DynabeadsUntouched magnetic separation kit (Invitrogen) or as previouslydescribed. CD4+ T cells were cultured in RPMI 1640 plus 10% fetal bovineserum (FBS) and 1× penicillin-streptomycin (Invitrogen). Resting CD4 Tcells were activated by culturing in PHA (2 μg/ml) plus IL-2 (2 ng/mL)(PepTech). HEK293T cells (ATCC) and HeLaJC.53 cells (NIH AIDS ReagentProgram) were maintained in Dulbecco-modified Eagle's medium (DMEM)(Invitrogen) containing 10% FBS and 1× penicillin-streptomycin(Invitrogen). PSGL-1-HeLaJC53 and Empty-HeLaJC53 cells were cultured inDMEM supplemented with 10% FBS and 550 μg/ml hygromycin B (Invitrogen).TZM-b1 cells (NIH AIDS Reagent Program) were cultured in DMEM containing10% FBS and 1× penicillin-streptomycin (Invitrogen). Jurkat cells (NIHAIDS Reagent Program) were cultured in RPMI 1640 supplemented with 2 mML-glutamine, 10% FBS and 1× penicillin-streptomycin (Invitrogen). HIVRev-dependent GFP indicator cells Rev-A3R5-GFP (Virongy) were culturedin RPMI 1640 plus 10% FBS supplemented with 1 μg/ml G418 (Sigma-Aldrich)and 1 μg/ml puromycin (Sigma-Aldrich). A3R5.7 cells (NIH AIDS ReagentProgram) were cultured in RPMI-1640 containing 10% FBS, 1% L-Glutamine,1× penicillin-streptomycin, and 1 μg/mL G418 (Invitrogen). CEM-SS cells(NIH AIDS Reagent Program) were cultured in RPMI-1640 with 10% FBS. Toconstruct PSGL-1-HeLaJC53 cells, HeLaJC.53 cells were seeded into a6-well plate and cultured in DMEM with 10% FBS. Cells were transfectedwith 2 μg pCMV3-PSGL-1 or pCMV3-Empty DNA using Jetprime transfectionreagent (Polyplus) as recommended by the manufacturer. Transfected cellswere cultured and selected with DMEM containing 10% FBS and 550 μg/ml ofhygromycin B (Invitrogen) to generate stably transfected cells.

Plasmids, Vectors, and Transfections

The infectious HIV-1 molecular clone pNL4-3, codon-optimized Vpuexpression vector (pcDNA-Vphu), Nef expression vector (pNef-ER), andNL4-3 ΔVpu infectious molecular clone (pNL-U35) were obtained from theNIH AIDS Reagent Program. pCMV3-PSGL-1 and pCMV3-Empty vectors wereobtained from Sinobiological. pNLΔΨEnv (gp160) and pHCMV-G expressingthe HIV-1 Env and the vesicular stomatitis virus G glycoprotein,respectively, were described previously. pNL4-3ΔNef was describedpreviously. The env-defective pNL4-3 derivative pNL4-3/KFS was describedpreviously.

The procedure for transfection of HEK293T cells to produce HIV-1particles was described previously. For transient transfection ofHeLaJC.53 cells, 0.5 million cells were transfected with 2 μg of eitherpCMV3-Empty or pCMV-PSGL-1 using the transfection reagent Jetprime(Polyplus) as recommended by the manufacturer. Following transfection,cells were cultured for the indicated times until analysis. For the p24release assay in HEK293T cells, cells were cotransfected with 1 μg ofHIV-1 NL4-3 and indicated doses of pCMV3-PSGL-1 or pCMV3-Empty DNA usingLipofectamine 2000 (Invitrogen). Supernatant was collected at 48 hoursposttransfection. To purify virions by ultracentrifugation, supernatantsharvested from transfected HEK293T cells were purified by ultra-speedcentrifugation through a gradient of 6-18% OptiPrep solution(Sigma-Aldrich), followed by a second round of ultracentrifugation usinga swinging-bucket rotor SW41Ti (Beckman) to pellet the virus (41,000rpm).

FACS Analysis

For PSGL-1 surface staining, 0.5-1 million cells were stained withanti-PSGL1 antibody [KPL-1] (BD Pharmingen) followed by staining withAlexa Fluor 488-conjugated goat anti-mouse secondary antibody(Invitrogen). For surface staining of infected blood resting CD4+ Tcells, HIV-1 infection was done using 125 ng to 320 ng p24 gagequivalents of NLHG1-ES-IRES-GPF reporter virus per million cells. Cellswere washed and cultured in 10% FBS RPMI with IL-7 (2 ng/mL). On theindicated days, cells were harvested and stained at 4° C. for 30 minwith AF687 anti-PSGL-1 antibody (KPL-1, BD Pharmingen) and analyzed byflow cytometry. For surface PSGL-1 staining of Jurkat, CEM-SS, andA3R5.7 cells, 0.5 million cells were stained with FITC-conjugatedanti-PSGL-1 antibody (Abcam) and analyzed by flow cytometry. ForHIV-1-infected Jurkat T-cell surface staining, 0.5 million cells wereinfected with different volumes of HIV-1 NL4-3. At 3 days postinfection, cells were stained with anti-PSGL-1 antibody [KPL-1] (BDPharmingen), followed by staining with Alexa Fluor 488-conjugated goatanti-mouse secondary antibody (Invitrogen) and flow cytometry analysis.For HEK293T cells, 0.5 million cells were cotransfected with differentdosages (1 μg to 4 μg) of HIV NL4-3 Vpu or HIV NL4-3 Nef, and 100 ng ofpCMV3-PSGL-1 using Lipofectamine 2000 (Invitrogen). Cells were stainedat 48 hours posttransfection with anti-PSGL-1 antibody [KPL-1] (BDPharmingen), followed by staining with Alexa Fluor 488-conjugated goatanti-mouse secondary antibody (Invitrogen).

Western Blots to Detect PSGL-1 and HIV-1 Proteins

The following antibodies were from the NIH AIDS Reagent Program:anti-HIV-1 p24 monoclonal antibody (183-H12-5C), anti-HIV Env (16H3)antibody, anti-HIV-1 gp41 monoclonal antibody (2F5), anti-HIV-1 gp41monoclonal antibody (10E8), and anti-HIV immune globulin (HIVIG). Cellsor virus pellets were solubilized in lysis buffer containing 50 mMTris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, andprotease inhibitor cocktail (Roche Life Science, Basel, Switzerland) orLDS lysis buffer (Invitrogen). Proteins were denatured by boiling insample buffer and subjected to SDS-PAGE, transferred to PVDF ornitrocellulose membrane, and incubated overnight at 4° C. with one ofthe following primary antibodies: anti-PSGL-1 (clone KPL-1, BDPharmingen) (1:1000 dilution); anti-PSGL-1 TC-2 (Abcam) (1:1000dilution); anti-PSGL-1 polyclonal (Abcam) (1:1000 dilution); anti-GAPDHgoat polyclonal antibody (Abcam) (1:1000 dilution); anti-CD45RA antibody(BD Biosciences); or HIVIG; 183-H12-5C, 16H3, 2F5, 10E8, HIVIG.Membranes were then incubated with HRP-labeled goat anti-mouse IgG (KPL)(1:2500 dilution) or anti-rabbit IgG (Cell Signaling) (1:2000 dilution)for 60 min at RT. Chemiluminescence signal was detected by using WestPico or West Femto chemiluminescence reagent (Thermo Fisher Scientific).Images were captured with a CCD camera (FluorChem 9900 Imaging Systems)(Alpha Innotech). Protein bands were also quantified usingImagelab-Chemidoc (Bio-Rad Laboratories, France). On some occasions,western blot was also performed using infrared imaging (Odyssey infraredimager, LI-cor Biosciences) with IRDye goat anti-mouse or rabbit 680 or800 cw labeled antibodies (Li-cor Biosciences) (1:5000 diluted inblocking buffer) for 1 h at 4° C. The blots were washed three times for15 minutes and scanned with Odyssey Infrared Imager (Li-corBiosciences). The ratios of gp120/p24 and gp160/p24 were quantified invirions, and the ratio of gp120/gp160 and expression of PSGL-1 werequantified in cell and virus fractions. To quantify virus releaseefficiency, HEK293T cells were transfected with the indicated plasmids(WT pNL4-3, pNL434Δpu, or pNL43ΔNef) in the absence or presence ofPSGL-1 expression vector using Lipofectamine 2000 (Invitrogen) orpolyethylenimine (PEI) transfection reagent (Sigma-Aldrich). At 30 to 48hours after the addition of DNA, virus-containing supernatant washarvested for p24 ELISA, or filtered and pelleted in an ultracentrifugefor analysis. The viral release efficiency (VRE) was calculated as theamount of virion-associated Gag as a fraction of total (cell- andvirion-associated) Gag quantified from Western blot analysis.

p24 ELISA

HIV-1 p24 released into the cell culture supernatant was detected by anin-house p24 ELISA kit. Briefly, microtiter plates (Sigma-Aldrich) werecoated with anti-HIV-1 p24 monoclonal antibody (183-H12-5C) (NIH AIDSReagent Program). Samples were incubated for 2 hours at 37° C., followedby washing and incubating with biotinylated anti-HIV immune globulin(HIVIG) (NIH AIDS Reagent Program) for 1 hour at 37° C. Plates were thenwashed and incubated with avidin-peroxidase conjugate (Thermo FisherScientific) for 1 hour at 37° C., followed by washing and incubatingwith tetramethylbenzidine (TMB) substrate. Plates were kinetically readusing an ELx808 automatic microplate reader (Bio-Tek Instruments) at 630nm.

Viral Entry Assay (BLAM Assay)

The viral entry assay was performed as previously described. Briefly,viruses were generated by co-transfection of HEK293T cells with threeplasmids: pNL4-3, pAdvantage (Promega) and pCMV4-3BlaM-Vpr (kindlyprovided by Dr. Warner C. Greene) (in a ratio of 6:1:2). Supernatant washarvested at 48 hours posttransfection, concentrated, and then used forinfection as suggested. Flow cytometry was performed using a BectonDickinson LSR II (Becton Dickinson). β-lactamase and CCF2 measurementswere performed using a 407-nm violet laser with emission filters of525/50 nm (green fluorescence) and 440/40 nm (blue fluorescence),respectively. Green and blue emission spectra were separated using a505LP dichroic mirror. The UV laser was turned off during the analysis.Data analysis was performed using FlowJo software (FlowJo).

Viral Attachment Assay

Virion particles produced in the presence of PSGL-1 or the empty vectorwere incubated with HelaJC53 cells at 4° C. for 2 hours. The cells werethen washed extensively (5 times) with cold PBS buffer and then lysedwith LDS lysis buffer (Invitrogen) for analysis by Western blot.

Infectivity Assays

For flow cytometry-based infectivity assay, virus particles wereproduced in HEK293T cells by cotransfection with pNL43, pNL43ΔVpu, orpNL43ΔNef with pCMV3-PSGL1 or pCMV3-Empty, or by cotransfection withpNL4-3/KFS, pHCMV-G, and pCMV3-PSGL-1 or pCMV3-Empty vector (using theindicated plasmid inputs) in a 6-well plate with Lipofectamine 2000(Invitrogen). Rev-A3R5-GFP cells were infected with each of theindicated viruses (0.5 million cells/infection). The cells were thenwashed and cultured in fresh media. Flow cytometry analysis of GFPexpression was performed on the indicated days. The percentage of GFP+cells was quantified.

For luciferase-based, single-cycle infectivity assays, RT-normalizedvirus stocks were used to infect the CD4+/CXCR4+/CCR5+ HeLa derivativeTZM-b1. This indicator cell line contains integrated copies of theβ-galactosidase and luciferase genes under the control of the HIV-1 LTR.Infection efficiency was determined by measuring luciferase activity 2days postinfection. For infectivity assays in HeLa JC53-PSGL-1 andHeLa-JC53-empty cell lines, the cells were seeded in 6-well plates at adensity of 0.2×106/well 24 hours prior to infection. Cells were infectedwith the indicated p24 equivalents of either WT NL4-3, NL43ΔVpu, orNL43ΔNef. Viral replication was quantified by virion p24 released intothe medium by p24 ELISA.

The embodiments describe a new approach to producing vaccines comprisinglive attenuated, inactivated, or non-infectious virion particles, suchas HIV particles.

Other embodiments are also within the scope of the following claims.

What is claimed is:
 1. An in vitro method, comprising: (i) expressingP-selectin glycoprotein ligand-1 (PSGL-1) or a PSGL-1 mutant in humanimmunodeficiency virus (HIV) producing cells; and (ii) isolating HIVparticles from the HIV producing cells; wherein the HIV particlescomprise non-infectious HIV particles, attenuated HIV particles orinactivated HIV particles; wherein the PSGL-1 or the PSGL-1 mutant is anoverexpressed PSGL-1 or an overexpressed PSGL-1 mutant; and wherein theoverexpressed PSGL-1 or the overexpressed PSGL-1 mutant is made by arecombinant technique.
 2. The in vitro method of claim 1, wherein themethod does not require a chemical that changes a structure of the HIVproducing cells.
 3. The in vitro method of claim 1, wherein the PSGL-1or the PSGL-1 mutant is overexpressed in the HIV producing cells.
 4. Thein vitro method of claim 1, wherein the HIV particles are thenon-infectious HIV particles.
 5. The in vitro method of claim 1, whereinthe HIV particles are the attenuated HIV particles.
 6. The in vitromethod of claim 1, wherein the HIV particles are the inactivated HIVparticles.
 7. The in vitro method of claim 1, wherein the PSGL-1 isexpressed by introducing a vector expressing the PSGL-1 or a PSGL-1mutant into the HIV producing cells.
 8. The in vitro method of claim 1,wherein the HIV particles are virions; wherein the virions arenon-infectious virion particles.
 9. The in vitro method of claim 8,wherein the non-infectious virion particles do not attach to target CD4T cells, the non-infectious virion particles are endocytosed by anantigen presenting cell to stimulate an anti-HIV humoral immunity. 10.The in vitro method of claim 8, wherein the PSGL-1 interfere withorganization of proteins within the virions.
 11. An in vitro method,comprising expressing PSGL-1 or a PSGL-1 mutant in virus producingcells; and inhibiting the virus producing cells from producing aninfectious virus particles; wherein the virus producing cells producenon-infectious virion particles; wherein the PSGL-1 or the PSGL-1 mutantis an overexpressed PSGL-1 or an overexpressed PSGL-1 mutant; andwherein the overexpressed PSGL-1 or the overexpressed PSGL-1 mutant ismade by a recombinant technique.
 12. The in vitro method of claim 11,wherein the virus producing cells are a host cell infected with a virus.13. The in vitro method of claim 12, wherein the virus is HIV.
 14. Thein vitro method of claim 11, wherein the virus producing cells producenon-infectious virion particles.
 15. An in vitro method, comprising:expressing PSGL-1 or a PSGL-1 mutant in virus producing cells; allowingthe virus producing cells to produce virions; isolating the virions; andpreparing non-infectious virions wherein the non-infectious virions areconfigured to elicit an immune response in a subject; and whereinexpressed PSGL-1 or expressed PSGL-1 mutant is made by a recombinanttechnique.
 16. The in vitro method of claim 15, wherein the virions areHIV particles.
 17. The in vitro method of claim 15, wherein the virionsare lentivirus particles.
 18. The in vitro method of claim 15,comprising treating a disease state in the subject resulting from avirus infection.
 19. The in vitro method of claim 1, wherein the HIVparticles comprise a human immunodeficiency virus (HIV) particle withdeleted Vpu.